Type 2 diabetes mellitus (T2DM) is characterized by a progressive loss of b-cell function associated with obesity. There is a fundamental gap in understanding how chronic nutrient overload associated with obesity alters human b-cell function. Elucidation of nutrient-induced changes in b-cell physiology may provide insight that leads to the development of pharmaceutical approaches to preserve b-cell function and mass in susceptible obese individuals. The objective of the proposed research is to elucidate the alterations in b-cell metabolic signaling under chronic nutrient overload and their role in b-cell defects. The central hypothesis is that mammalian target of rapamycin complex 1 (mTORC1), a nutrient sensor, plays a pivotal role in two major metabolic alterations: ectopic lipid accumulatio and islet cell expansion. The hypothesis will be tested by pursuing two specific aims: 1) Elucidate the signaling pathways that lead to nutrient-mediated ectopic lipid accumulation and islet cell expansion; and 2) Determine the role of ectopic lipid accumulation, peroxynitrite, and islet cell expansion in b-cell function and survival. Under the first aim, molecular mechanisms involved in b-cell metabolic perturbations will be delineated using newly established microscopic methods and biochemical assays. The molecular mechanisms will be probed with rapamycin, an inhibitor of mTORC1; BN99, an inhibitor of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the final step of triglyceride (TG) biosynthesis; and SR-135, a peroxynitrite decomposing catalyst. Under the second aim, the causal relationship between adaptive responses and b-cell function and survival will be investigated by measuring insulin secretion, insulin content, and b-cell apoptosis under the conditions of excess nutrients in the presence and absence of rapamycin, BN99, or SR-135. Quantitation of potentially toxic compounds such as free fatty acids, ceramide, diacylglycerol, and other intermediates of the TG esterification pathway will supplement these results. The approach is highly innovative because new microscopic methodologies for the simultaneous assessment of multi-factorial b-cell responses to metabolic perturbations are utilized. The proposed research is significant because it will have an important positive impact on the development of new targets for preventive and therapeutic interventions for T2DM in addition to fundamentally advancing the field of the human islet biology vertically. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because understanding of the human b-cell metabolic signaling under chronic nutrient overload is expected to have an important positive impact on the development of new targets for preventive and therapeutic interventions for type 2 diabetes. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help to reduce the burdens of human disability.